Benefits derived from the measurement of oxygen concentrations in tissue are known. Oxygen is the primary biological oxidant, and the measurement of oxygen tension pO.sub.2 (positive pressure of oxygen) can improve the evaluation and understanding of many physiological, pathological, and therapeutic processes.
Prior art systems and methods for measuring oxygen concentrations in tissue are also known, including: the Clark electrode, fluorescence quenching, O.sub.2 binding to myoglobin and hemoglobin, chemiluminescence, phosphoresence quenching, and spin label oximetry. However, these systems and methods have certain, and often acute, limitations, especially when used in vivo. They especially lack the qualities required for complete experimental and clinical use, such as sensitivity, accuracy, repeatability, and adequate spatial resolution. See J. Chapman, Radiother. Oncol. 20, 13 (1991) and J. M. Vanderkooi et al., "Oxygen in Mammalian Tissue: Methods of Measurement and Affinities of Various Reactions", Am. J. Physiol. 260, C1131 (1991).
The polarographic microelectrode is one popular device for measuring oxygen tension in tissue. However, it has obvious technical difficulties associated with the repeated insertion of the microelectrode into the tissue. For example, the microelectrode often damages the tissue, and there is repeated difficulty in re-positioning the microelectrode at the same test location. The microelectrode is also relatively insensitive to oxygen concentrations below 10 mm Hg, which is within the required sensitivity region for effective oximetry. Finally, the microelectrode may itself consume oxygen, thereby altering its own environment, inducing measurement errors, and reducing the accuracy and usefulness of the evaluation process.
There are scattered reports which concern in vivo pO.sub.2 measurements with such devices, especially in skeletal muscle. Whalen and Nair, Am. J. Physiol. 218, 973 (1970), measured pO.sub.2 of cat gracilis at rest using a recessed Au 1-51 .mu.m microelectrode, giving average pO.sub.2 values of 6.6.+-.0.4 mm Hg (n=372). Gayeski et al., Am. J. Physiol. 254, H1179 (1988), measured pO.sub.2 of dog gracilis at rest, exhibiting a partial pressure range of 4.5-35 mm Hg (16.8 mm Hg median), and 95% VO.sub.2 max, using a Mb saturation technique, exhibiting a partial pressure range of 0.2-2.3 mm Hg (0.9-1.8 range of mean). Nevertheless, there are effective limitations to these pO.sub.2 measurement techniques. In the microelectrode method, for example, it is technically difficult to monitor or make long term evaluations of pO.sub.2. In the Mb saturation method, it is especially difficult to measure low pO.sub.2, and the method can only be used in muscle.
Nuclear Magnetic Resonance (NMR) techniques have been explored and considered in the context of oxiometric measurements, especially through the use of an oxygen dependent proton hyperfine line in myoglobin and oxygen dependent relaxation of fluorine nuclei. NMR is a common spectroscopic technique in which the molecular nuclei is aligned in a magnetic field and simultaneously excited by absorption of radiofrequency energy. The molecular relaxation from the excited state to the initial state is an observable event that is affected by the presence of oxygen through exchange or dipolar actions. However, the NMR techniques have not demonstrated sufficient sensitivity and/or applicability to the measure of pO.sub.2 in either experimental or clinical settings.
Electron Paramagnetic Resonance (EPR) oximetry is another technique for measuring oxygen tension. Similar to NMR, EPR oximetry is a spectroscopic technique based upon the Zeeman effect and the line-broadening effect of molecular oxygen on the EPR spectra of paramagnetic materials. These materials have unpaired electron spins that are aligned in a magnetic field and excited by microwave energy. The separation between the lower, unexcited energy state and the higher, excited energy state is proportional to the strength of the magnetic field. The presence of oxygen with the excited molecule measurably affects the molecular relaxation so that the line width of the EPR spectra changes and provides an indication of pO.sub.2.
Nitroxides exemplify one family of compounds having paramagnetic quality that are suitable for EPR oximetry, and which have been used in a variety of in vitro experiments. Although nitroxides have also been tested in vivo, at least two resulting problematic areas exist in such measurements: first, nitroxides tend to be bioreduced; and secondly, nitroxides are relatively insensitive to low oxygen tension levels that are of the most biological interest today, i.e., less than 10 mmHg.
Other recent discoveries of new paramagnetic materials, such as Fusinite and lithium phthalocyanine (LiPc), have made progress as oxygen probes in the field of in vivo EPR oximetry. These two compounds, for example, are suitable for in vivo usage because they exhibit certain favorable characteristics, including: accuracy; spatial resolution; sensitivity in the physiologically important oxygen tension range; ease of use; little or no apparent toxicity; and relative stability in tissues, permitting prolonged measurements over periods of weeks or months after administering the compound. Nevertheless, because these paramagnetic compounds have not been previously tested in humans, they will have to undergo very long and extensive toxicological evaluation before they can be used clinically. This evaluation is likely to be prolonged because of other problems inherent in the compounds, such as stability and inertness, which encourage indefinite, unwanted persistence within the tissue.
There are other existing problems limiting the effectiveness of EPR oximetry, including the inability to measure EPR spectra efficiently and effectively, especially in vivo. Conventional EPR spectrometers, for example, typically utilize microwave frequencies, e.g., 9 GHz, that are strongly absorbed by tissue and water, and which reduce the useful depth penetration and measurement sensitivities within the tissue. Prior EPR spectrometers also cannot effectively measure EPR spectra from a biological system such as a live animal, because movements of the animal change the observed EPR spectra. This movement increases noise and reduces the accuracy. Finally, conventional EPR spectrometers have the resonator and the sample under test, e.g., tissue, within a common magnetic field. This constrains the EPR measurement/flexibility, being subject to physical size considerations, and potentially to the patient's dexterity.
It is accordingly an object of this invention to provide an improved EPR spectrometer and associated methodology that are free of the afore-mentioned difficulties.
It is another object of this invention to provide an improved apparatus and method that enables the direct measurement of oxygen concentration in biological systems, such as tissue.
It is a further object of the invention to provide improved methodology and apparatus for in vivo EPR oximetry.
Other objects of the invention will be apparent from the following description.